The Golgi apparatus is a membranous organelle that plays a pivotal role in protein post-translational modification, sorting, and transport. In interphase animal cells, the Golgi apparatus manifests a crescent moon shaped, ribbon-like morphology in the perinuclear region, normally situated closely with centrosomes.
Microtubule cytoskeleton plays an essential role in the organization, positioning, and function of the Golgi apparatus (Rios and Bornens, Curr. Opin. Cell Biol. 15:60-66 (2003); Sutterlin and Colanzi, J. Cell Biol. 188:621-628 (2010); Lippincott-Schwartz, Curr. Opin. Cell Biol. 10:52-59 (1998)). Microtubule depolymerization causes severe Golgi defects, such as endoplasmic reticulum (ER)-to-Golgi transport blockage and Golgi fragmentation (Cole et al., Mol. Biol. Cell. 7:631-650 (1996); Miller et al., Nat. Cell Biol. 11:1069-1080 (2009)). The Golgi apparatus undergoes disassembly and reassembly during mitosis. During reassembly, microtubules derived from the Golgi apparatus and centrosomes facilitate the Golgi apparatus to form into a continuous ribbon structure with the central cellular positioning from Golgi ministacks (Miller et al., Nat. Cell Biol. 11:1069-1080 (2009)).
The Golgi apparatus serves as a major microtubule-organizing center (Chabin-Brion et al., Mol. Biol. Cell. 12:2047-2060 (2001); Efimov et al., Dev. Cell. 12:917-930 (2007); Miller et al., Nat. Cell Biol. 11:1069-1080 (2009); Rivero et al., EMBO J. 28:1016-1028 (2009)). In humans, almost half of cellular microtubules originate from the Golgi apparatus as observed in retinal pigment epithelial cells RPE1 (Efimov et al., Dev. Cell. 12:917-930 (2007)). Golgi-associated microtubules are also required for Golgi ribbon assembly, directional trafficking, and cell motility (Miller et al., Nat. Cell Biol. 11:1069-1080 (2009); Rivero et al., EMBO J. 28:1016-1028 (2009)). Microtubule nucleation at the Golgi apparatus does not require centrosomes; rather, it depends on γ-tubulin (Efimov et al., Dev. Cell. 12:917-930 (2007)), the principal microtubule nucleator that exists as γ-tubulin complexes (γTuCs). γTuCs associate with two cis-Golgi proteins, GMAP-210 and AKAP450 (AKAP450 is also known as AKAP350, CG-NAP, and hyperion) (Rios et al., Cell 118:323-335 (2004); Rivero et al., EMBO J. 28:1016-1028 (2009); Takahashi et al., Mol. Biol. Cell 13:3235-3245 (2002)). It is reported that silencing AKAP450 expression blocks the Golgi-associated microtubule nucleation (Rivero et al., EMBO J. 28:1016-1028 (2009)).
Golgi-nucleated microtubules are required for post-Golgi secretion and directional cell migration. In the secretory pathway, protein transport from the ER to the Golgi apparatus is initiated by cargo packaging into COPII-coated vesicles at ER exit sites, followed by the formation of vesicular-tubular clusters or the ER-Golgi intermediate compartment that move along microtubules towards the Golgi apparatus. Defects of protein secretion can cause a number of diseases, including neuronal disorders and diabetes. In addition, as microtubules are involved in cell migration, suppression of microtubule nucleation may be used to inhibit tumor metastasis.
EB1 is the prototypic member of EB proteins, which are localized to microtubules and track growing microtubule plus-ends (Akhmanova and Steinmetz, Nat. Rev. Mol. Cell Biol. 9:309-322 (2008); Vaughan, J. Cell Biol. 171:197-200 (2005)). Among the EB proteins, EB1 and EB3 display similar tip-tracking properties, whereas EB2 appears to be distinct from the other two, exhibiting a considerably weaker tip-tracking activity (Komarova et al., J. Cell Biol. 184:691-706 (2009)).
EB1 is found on all growing microtubule tips, where it acts as a key component of plus-end protein complexes through its interaction with various plus-end tracking proteins (+TIPs). Many +TIPs contain the SxIP motif surrounded by basic and serine-rich sequences for interaction with the EBH domain of EB1 (Honnappa et al., Cell 138:366-376 (2009)). This SxIP-EB1 interaction relieves EB1 autoinhibition, and is required for EB1 in its action for promoting microtubule polymerization (Honnappa et al., EMBO J. 24:261-269 (2005); Honnappa et al., Cell 138:366-376 (2009); Slep et al., J. Cell Biol. 168:587-598 (2005)). +TIPs in association with EB1 at the microtubule plus-ends have diverse functions, including regulation of microtubule dynamics and microtubule attachment to subcellular targets (Akhmanova and Steinmetz, Nat. Rev. Mol. Cell Biol. 9:309-322 (2008)).
In a yeast two-hybrid screen, myomegalin (MMG) isoform 1 (Genbank accession: NP—055459) was cloned to interact with cyclic nucleotide phosphodiesterase 4D, and was identified as a phosphodiesterase 4D-interacting protein (Verde et al., J. Biol. Chem. 276:11189-11198 (2001)). The MMG1 nucleotide sequence encodes a ˜230 kDa protein that is highly expressed in heart and skeletal muscles. In gene databases, MMG1 is the only known human homolog of CDK5RAP2 (CDK5 regulatory subunit-associated protein 2), a human microcephaly-related protein involved in microtubule organization in centrosomes as well as microtubule regulation at the growing tips (Fong et al., Mol. Biol. Cell. 19:115-125 (2008); Fong et al., Mol. Biol. Cell. 20:3660-3670 (2009)). MMG isoform 1 displays centrosome and Golgi-localizing patterns similar to CDK5RAP2 (Verde et al., J. Biol. Chem. 276:11189-11198 (2001); Wang et al., J. Biol. Chem. 285:22658-22665 (2010)).
Despite the apparent importance of the Golgi apparatus in various cellular activities such as protein modification and trafficking, microtubule organization, and cell migration, the precise mechanisms of how the Golgi apparatus exerts these functions have not been fully elucidated. Specifically, it remains unclear how γTuCs are targeted to the Golgi apparatus. In addition, it remains unclear whether EB1 and other EB members are present at the Golgi apparatus to exert their functions. As will be clear from the disclosures that follow, these and other benefits are provided by the present invention.